Concerted use of restriction endonucleases and DNA ligases allows in vitro recombination of DNA sequences. The recombinant DNA generated by restriction and ligation may be amplified in an appropriate microorganism such as E. coli, and used for diverse purposes including gene therapy. However, the restriction-ligation approach has two practical limitations: first, DNA molecules can be precisely combined only if convenient restriction sites are available; second, because useful restriction sites often repeat in a long stretch of DNA, the size of DNA fragments that can be manipulated is limited, usually to less than about 25 kilobases.
Homologous recombination, generally defined as an exchange between homologous segments anywhere along a length of two DNA molecules, provides an alternative method for engineering DNA. In generating recombinant DNA with homologous recombination, a microorganism such as E. coli, or a eukaryotic cell such as a yeast or vertebrate cell, is transformed with exogenous DNA. The center of the exogenous DNA contains the desired transgene, whereas each flank contains a segment of homology with the cell's DNA. The exogenous DNA is introduced into the cell with standard techniques such as electroporation or calcium phosphate-mediated transfection, and recombines into the cell's DNA, for example with the assistance of recombination-promoting proteins in the cell.
A recombination system (termed “recombineering) has been developed for efficient chromosome engineering in Escherichia coli using electroporated linear DNA (see published PCT Application No. WO 02/14495 A2, which is herein incorporated by reference). A defective prophage supplies functions (λ Red) that protect and recombine an electroporated DNA substrate in the bacterial cell. This system can be used with single-stranded DNA, as well as with linear double-stranded DNA (dsDNA). The use of recombination eliminates the requirement for standard cloning as all novel recombination sites are engineered by chemical synthesis in vitro, and the linear DNA is efficiently recombined in vivo. In this system, a temperature-dependent repressor tightly controls prophage expression, such that recombination functions can be transiently supplied by shifting cultures to 42° C. The efficient prophage recombination system does not require host RecA function and depends primarily on exo, bet, and gam functions expressed from the defective prophage. The defective prophage can be moved to other strains and can be easily removed from any strain. Importantly, recombination in this system is proficient with DNA homologies as short as 30-50 base pairs, making it possible to use PCR-amplified fragments as the targeting cassette. Gene disruptions and modifications of both the bacterial chromosome and bacterial plasmids are possible, and the system has been shown to be of use in the bacterial artificial chromosome libraries (see Published PCT Application No. WO 02/14495, herein incorporated by reference; Yu et al., Proc. Natl. Acad. Sci. USA 97:5978-5983, 2000).
This prophage system has been adapted for use in bacterial artificial chromosome (BAC) engineering by transferring it to DH10B cells, a BAC host strain. Fragments as large as 80 kb can be subcloned from BACs by gap repair using this recombination system, obviating the need for restriction enzymes or DNA ligases. BACs can be modified with this recombination system in the absence of drug selection (Lee et al., Genomics 73:56-65, 2001). It has been suggested that recombineering in BACs allows modification or subcloning of large fragments of genomic DNA with precision. This ability facilitates many kinds of genomic experiments that were difficult or impossible to perform previously and aid in studies of gene function. It has been suggested that this system is of use in generating mouse models and providing a refined analysis of the mouse genome (Copland et al., Nat. Rev. Genet. 2:769-779, 2001).
Recombineering uses the exo and bet functions of the prophage lambda under the control of a temperature sensitive repressor. When the lambda functions are turned on, cells become more “recombinogenic,” that is they take up DNA and recombination of the DNA occurs with a target sequence in the cell. This system has been adapted for use in bacterial artificial chromosome engineering, wherein inducible recombinases (e.g. cre or flpe) are introduced into host cells and BAC modification is accomplished using recombination sites (e.g. loxP or frt, respectively). This system can be used to generate Cre-expressing transgenic mice for use in conditional knock-out studies. This system utilizes a targeting vector to introduce recombination sites (e.g. loxP) into a gene of interest. Expression of a recombinase from a specific promoter (such that expression occurs in a tissue of interest) results in recombination at the recombination sites, leading to a “conditional knock-out.”
It has also been shown that synthetic single-stranded oligonucleotides (SSOs) can also be used in place of linear dsDNA fragments, to create sequence-specific mutations (Ellis et al., Proc. Natl Acad Sci USA 98:6742-6746, 2001; Swaminathan et al., Genesis 29:14-21, 2001). However, the mechanism(s) by which SSOs affect these genetic changes remains to be established. In addition, although recombineering using SSOs provides a method for introducing homologous DNA into a target nucleic acid sequence, the frequency of recombination can be improved. Thus, methods for increasing recombination frequency using recombineering, and strains which produce recombinants at a high frequency are disclosed herein.